Rapid immunochromatographic assay devices (also referred to herein simply as “rapid assay devices”) are currently available to test clinical samples (e.g. whole blood, serum, plasma, urine, saliva) for a wide variety of analytes, such as hormones, drugs, toxins, metabolites, cardiac markers, and pathogen-derived antigens. In addition, rapid assay devices are also used extensively in non-clinical applications such as food and environmental testing. Typical devices are comprised of an immunochromatographic assay strip contained within a housing that exposes selective portions of the strip, while at the same time concealing the majority of this strip component. FIG. 1A shows a typical device 10 that includes a plastic housing 12, containing an assay strip, which is accessed through a sample receiving port 14, and viewable through one or more windows that expose the test zone 16, and the control zone 18. FIG. 1B shows the position of the assay strip 11 within the housing. In an exemplary implementation of the device, a fluid sample is applied to the sample receiving port and a period of time is allowed to elapse before viewing the results in the test and control windows. FIG. 1C shows a typical test result, in which a visible line forms inside the test and control windows (13 and 15, respectively). The band forms from a reaction process that occurs following application of sample to the device. This process typically creates in interim discoloration on the strip prior to the final viewed result. This interim state is disregarded with respect to interpreting the result. Indeed, many test protocols direct the user to avoid observing the interim discolored state to prevent difficulties with interpreting the final result.
Most rapid assay devices are designed for simple qualitative analysis, indicating either the presence or absence of an analyte at a particular cut-off level, based on a visual interpretation of band formation. For sandwich assays, the presence of an analyte in a sample is indicated by the formation of a line in the test zone, whereas for competition assays the presence of an analyte in a sample is indicated by the absence of a line forming in the test zone. The presence of the control line typically indicates that the assay has been correctly performed to completion. Inspection of the test and control zones occurs only after sufficient time has elapsed to allow for optimal viewing (typically 5-10 minutes following sample application). During this incubation period an immunochromatographic reaction is initiated, propagated and completed by the fluid sample migrating through the assay strip (via capillary action) and interacting with a series of reagents bound reversibly or irreversibly to the strip. Such reagents may include an analyte-specific binding pair (e.g. an antibody or antigen) coated onto labeled test particles, and an analyte-specific binding pair coated within the test zone.
Because rapid assay devices are simple to perform and can be interpreted visually without the aid of instrumentation, they are widely used for obtaining quick test results outside of laboratory settings (usually at the site of sample collection), thus providing a convenient alternative to transporting these samples to a laboratory for analysis. However, the advantages of these devices are offset by the fact that they are considerably less reliable than alternative laboratory-based immunoassays, and generally unable to provide quantitative results. Laboratory immunoassays (such as those performed on automated analyzers or with ELISA kits) incorporate precisely defined and controlled assay conditions. These conditions involve such aspects as the concentration or molar ratio of sample components to reagent components, the reaction volumes, and the reaction incubation times. Calibrator and control samples are also incorporated as part of the standard laboratory protocol and performed under the same conditions as the test samples. Deviation from the defined assay conditions can result in erroneous test results. In a laboratory setting this deviation is avoided with the use of sophisticated instrumentation, trained personnel and strict operating procedures.
In contrast to laboratory-based immunoassays, rapid assay devices are highly limited in their ability to deliver defined assay conditions, as these conditions are dictated by various flow dynamics that cannot be precisely replicated on each device. The assay reaction is induced by the application of fluid sample onto the strip, where it initially encounters reversibly bound test particles. As capillary action moves the sample through the strip, the test particles are rehydrated and mobilized. The rate at which the particles mobilize, total number of particles mobilized, and direction of particle migration across the strip, collectively contribute to the concentration and molar ratio of active reagent molecules to analyte molecules. As the sample and particles continue to flow along the strip, they eventually come in contact with the test zone where a second set of reagents is immobilized. The time required to reach the test zone, and the rate at which the sample and particles flow through the test zone, effectively define the assay incubation times. Thus, the assay conditions of a typical rapid assay device are largely governed by flow dynamics, which are in turn governed by properties of the test device that cannot be precisely reproduced for each individual device, resulting in device-to-device variation in assay conditions. Such properties of the device include membrane porosity, contact forces between membranes, and adhesion forces between the membrane and embedded test particles.
Attempts have been made to improve rapid test reliability by incorporating photo-optic reading instruments that measure and analyze color intensity of the test band. While this approach allows the test band to be analyzed with greater objectivity and quantitation (compared with visual interpretation), it fails to address the underlying problem of variable flow dynamics that can non-specifically influence the intensity of test band formation. Other approaches have focused on using the intensity of the control line to normalize the results of the test band. This approach does incrementally improve reliability and quantitation; however, the results still remain far inferior to laboratory-based systems. In addition, the control line approach requires considerably greater manufacturing effort compared to that of a standard rapid assay device and introduces additional variables that can compromise the interpretation of test results.
There remains a compelling need to develop a rapid assay system that can be performed on-site (outside of a laboratory setting) yet provide results with reliability and quantitation comparable to a laboratory-based system. The current invention is based on the surprising finding that reliable and quantitative rapid assays, suitable for on-site applications, can be developed using immunochromatographic components, despite the fact that assay conditions cannot be precisely controlled with such components.